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New imidazo[1,2-a]pyrazine derivatives have been synthesized either by direct cyclization from pyrazines or by electrophilic substitutions. The presence of electron donating groups on position 8 greatly enhances the reactivity of the heterocycle towards such reactions on position 3 of the heterocycle. The activities of these derivatives in trachealis muscle relaxation and in inhibiting cyclic nucleotide phosphodiesterase (PDE) isoenzyme types III and IV have been assessed. All compounds demonstrated significantly higher relaxant potency than theophylline. All the derivatives were moderately potent in inhibiting the type IV isoenzyme of PDE but only those with a cyano group on position 2 were potent in inhibiting the type III isoenzyme.

While UK-93,928 (1-[[3-(6,9-dihydro-6-oxo-9-propyl-1H-purin-2-yl)-4-ethoxyphenyl] sulfonyl]-4-methylpiperazine; 5 nM-5 microM) was devoid of relaxant activity, benzafentrine, isoprenaline, levcromakalim and SCA40 (6-bromo-8-methylaminoimidazo[1,2-a]pyrazine-2-carbonitrile) each relaxed histamine (460 microM)-precontracted bovine isolated trachealis. Each of these relaxants was antagonised by a K+-rich (80 mM) medium. Except in the case of levcromakalim, nifedipine (1 microM) offset this antagonism. Charybdotoxin (100 nM) antagonised isoprenaline in a nifedipine-sensitive manner but did not antagonise SCA40 or benzafentrine. Iberiotoxin (100 nM) did not antagonise SCA40. Acting on tissue precontracted with carbachol, SCA40 potentiated isoprenaline but did not potentiate sodium nitroprusside. While levcromakalim (1 and 10 microM) induced hyperpolarisation, SCA40 (1 and 10 microM) induced little change in the membrane potential of bovine trachealis. In trachealis preloaded with 86Rb+, levcromakalim (1 and 10 microM) promoted efflux of the radiotracer while SCA40 (1 and 10 microM) had no effect. Tested as an inhibitor of isoenzymes of cyclic nucleotide phosphodiesterase, SCA40 was most potent against the type III, less potent against the type IV and least potent against the type I isoenzyme. It is concluded that neither inhibition of phosphodiesterase type V nor the promotion of BKCa channel opening explains the tracheal smooth muscle relaxant activity of SCA40. This compound relaxes bovine tracheal smooth muscle mainly by inhibiting phosphodiesterase isoenzyme types III and IV.

1. Inhalation of vanadium compounds, particularly vanadate, is a cause of occupational bronchial asthma. We have now studied the action of vanadate on human isolated bronchus. Vanadate (0.1 microM-3 mM) produced concentration-dependent, well-sustained contraction. Its -logEC50 was 3.74 +/- 0.05 (mean +/- s.e.mean) and its maximal effect was equivalent to 97.5 +/- 4.2% of the response to acetylcholine (ACh, 1 mM). 2. Vanadate (200 microM)-induced contraction of human bronchus was epithelium-independent and was not inhibited by indomethacin (2.8 microM), zileuton (10 microM), a mixture of atropine, mepyramine and phentolamine (each at 1 microM), or by mast cell degranulation with compound 48/80. 3. Vanadate (200 microM)-induced contraction was unaltered by tissue exposure to verapamil or nifedipine (each 1 microM) or to a Ca2+-free, EGTA (0.1 mM)-containing physiological salt solution (PSS). However, tissue incubation with ryanodine (10 microM) in Ca2+-free, EGTA (0.1 mM)-containing PSS reduced vanadate-induced contraction. A series of vanadate challenges was made in tissues exposed to Ca2+-free EGTA (0.1 mM)-containing PSS with the object of depleting intracellular Ca2+ stores. In such tissues cyclopiazonic acid (CPA; 10 microM) prevented Ca2+-induced recovery of vanadate-induced contraction. 4. Tissue incubation in K+-rich (80 mM) PSS, K+-free PSS, or PSS containing ouabain (10 microM) did not alter vanadate (200 microM)-induced contraction. Ouabain (10 microM) abolished the K+-induced relaxation of human bronchus bathed in K+-free PSS. This action was not shared by vanadate (200 microM). The tissue content of Na+ was increased and the tissue content of K+ was decreased by ouabain (10 microM). In contrast, vanadate (200 microM) did not alter the tissue content of these ions. Tissue incubation in a Na+-deficient (25 mM) PSS or in PSS containing amiloride (0.1 mM) markedly inhibited the spasmogenic effect of vanadate (200 microM). 5. Vanadate (200 microM)-induced contractions were markedly reduced by tissue treatment with each of the protein kinase C (PKC) inhibitors H-7 (10 microM), staurosporine (1 microM) and calphostin C (1 microM). Genistein (100 microM), an inhibitor of protein tyrosine kinase, also reduced the response to vanadate. 6 Vanadate (0.1-3 mM) and ACh (1 microM- 3 mM) each increased inositol phosphate accumulation in bronchus. Such responses were unaffected by a Ca2+-free medium either alone or in combination with ryanodine (10 microM). 7. In human cultured tracheal smooth muscle cells, histamine (100 microM) and vanadate (200 microM) each produced a transient increase in intracellular Ca2+ concentration ([Ca2+]i). 8. Intracellular microelectrode recording showed that the contractile effect of vanadate (200 microM) in human bronchus was associated with cellular depolarization. 9. It is concluded that vanadate acts directly on human bronchial smooth muscle, promoting the release of Ca2+ from an intracellular store. The Ca2+ release mechanism involves both the production of inositol phosphate second messengers and inhibition of Ca-ATPase. The activation of PKC plays an important role in mediating vanadate-induced contraction at values of [Ca2+]i that are close to basal.

1. A study has been made of the effects of inhibitors selective among plasmalemmal K(+)-channels on the sensitivity and responsiveness of guinea-pig trachealis muscle to carbachol, histamine and KCl. The effects of the K(+)-channel inhibitors on the resting membrane potential and spontaneous electrical activity of the trachealis cells have also been examined. 2. In indomethacin (2.8 microM)-treated trachealis muscle, dofetilide (1 microM) and glibenclamide (10 microM) were each devoid of spasmogenic activity. In contrast, 4-aminopyridine (4-AP, 62.5 microM--8 mM), charybdotoxin (ChTX, 100 nM) and iberiotoxin (IbTX, 100 nM) were each spasmogenic. Spasm evoked by 4-AP, IbTX or ChTX was reduced, though not abolished, by atropine (1 microM). Spasm evoked by 4-AP (1 mM), ChTX (100 nM) or IbTX (100 nM) was unaffected by tetrodotoxin (TTX; 3.1 microM) or by tissue pretreatment with capsaicin (1 microM for 30 min). Spasm evoked by IbTX or ChTX was abolished by nifedipine (1 microM). 3. Dofetilide (1 microM) and glibenclamide (10 microM) were each without effect on the tracheal sensitivity or responsiveness to carbachol, histamine or KCl. 4-AP (1 mM) antagonized carbachol, potentiated histamine but did not affect tissue sensitivity to KCl. When the effects of 4-AP were examined in the presence of atropine (1 microM), it potentiated all the spasmogens including carbachol. IbTX and ChTX (each 100 nM) potentiated all three spasmogens. Potentiation of histamine induced by 4-AP (1 mM) or IbTX (100 nM) was also observed in tissues treated with a combination of atropine (1 microM) and TTX (3.1 microM). 4. Dofetilide (1 and 10 microM) was without effect on the resting membrane potential or spontaneous electrical activity of the trachealis cells. 4-AP (1 mM) evoked depolarization and caused a small increase in the frequency of slow wave discharge. The depolarization evoked by 4-AP was abolished by atropine (1 microM). IbTX (100 nM) and ChTX (100 nM) each evoked little or no change in resting membrane potential but converted the spontaneous slow waves into spike-like, regenerative action potentials. These electrophysiological effects of IbTX and ChTX were unaffected by atropine (1 microM). 5. It is concluded that the dofetilide-sensitive, cardiac, delayed rectifier K(+)-channel is either not expressed in trachealis muscle or is of no functional importance in that tissue. The ATP-sensitive K(+)-channel (KATP) does not moderate tracheal sensitivity to spasmogens such as carbachol, histamine and KCl. The 4-AP-sensitive delayed rectifier K(+)-channel (Kdr) and the large Ca(2+)-dependent K(+)-channel (BKCa) each moderate trachealis muscle sensitivity to spasmogens. Neither Kdr nor BKCa plays an important role in determining the resting membrane potential of guinea-pig trachealis cells. However, the BKCa channel is responsible for limiting the effects of the increase in membrane Ca2+ conductance associated with the depolarizing phase of slow waves. It is BKCa channel opening that prevents the development of a slow wave into a spike-like regenerative action potential.

1. SCA40 (1nM-10 microM), isoprenaline (1-300 nM) and levcromakalim (100 nM-10 microM) each produced concentration-dependent suppression of the spontaneous tone of guinea-pig isolated trachea. Propranolol (1 microM) markedly (approximately 150 fold) antagonized isoprenaline but did not antagonize SCA40. The tracheal relaxant action of SCA40 was unaffected by suramin (100 microM) or 8-(p)-sulphophenyltheophylline (8-SPT; 140 microM). 2. An isosmolar, K(+)-rich (80 mM) Krebs solution increased tracheal tone, antagonized SCA40 (approximately 60 fold), antagonized isoprenaline (approximately 20 fold) and very profoundly depressed the log concentration-effect curve for levcromakalim. Nifedipine (1 microM) did not itself modify the relaxant actions of SCA40, isoprenaline or levcromakalim. However, nifedipine prevented the rise in tissue tone and the antagonism of SCA40 and isoprenaline induced by the K(+)-rich medium. In contrast, nifedipine did not prevent the equivalent antagonism of levcromakalim. 3. Charybdotoxin (100 nM) increased tracheal tone, antagonized SCA40 (approximately 4 fold) and antagonized isoprenaline (approximately 3 fold). Nifedipine (1 microM) prevented the rise in tissue tone and the antagonism of SCA40 and isoprenaline induced by charybdotoxin. 4. Quinine (30 microM) caused little or no change in tissue tone and did not modify the relaxant action of isoprenaline. However, quinine antagonized SCA40 (approximately 2 fold). Nifedipine (1 microM) prevented the antagonism of SCA40 induced by quinine. 5. Tested on spontaneously-beating guinea-pig isolated atria SCA40 (1 nM-10 microM) increased the rate of beating in a concentration-dependent manner. Over the concentration-range 1 microM-10 microM, SCA40 also caused an increase in the force of atrial contraction. 6. Intracellular electrophysiological recording from guinea-pig isolated trachealis showed that the relaxant effects of SCA40 (1 micro M) were often accompanied by the suppression of spontaneous electrical slow waves but no change in resting membrane potential. When the concentration of SCA40 was raised to 10 micro M, its relaxant activity was accompanied both by slow wave suppression and by plasmalemmal hyperpolarization.7. SCA40 (10 nM- 100 micro M) more potently inhibited the activity of cyclic AMP phosphodiesterase (PDE)than that of cyclic GMP PDE derived from homogenates of guinea-pig trachealis. Theophylline(1 micro M- 1O mM) also inhibited these enzymes but was less potent than SCA40 in each case and did not exhibit selectivity for inhibition of cyclic AMP hydrolysis.8. Tested against the activity of the isoenzymes of cyclic nucleotide PDE derived from human blood cells and lung tissue, SCA40 proved highly potent against the type III isoenzyme. It was markedly less potent against the type IV and type V isoenzymes and even less potent against the isoenzymes types I and II.9. It is concluded that the tracheal relaxant action of SCA40 (1 nM- 1 micro M) does not involve the activation of beta-adrenoceptors or P1 or P2 purinoceptors. Furthermore, this action is unlikely to depend upon the opening of BKca channels with consequent cellular hyperpolarization and voltage-dependent inhibition of Ca2+ influx. The tracheal relaxant action of SCA40 (up to 1 micro M) is more likely to depend upon its selective inhibition of the type III isoenzyme of cyclic nucleotide PDE. At concentrations above 1 micro M, SCA40 exerts more general inhibition of the isoenzymes of cyclic nucleotide PDE and may then promote the opening of BKca channels.

1. Using guinea-pig isolated trachea, we have studied how phorbol 12,13-diacetate (PDA) modulates mechanical responses of the tissue to methylxanthines, isoprenaline and ryanodine. 2. Caffeine (10 microM-5 mM), theophylline (10 microM-5 mM) and isoprenaline (1 nM-1 microM), each inhibited the spontaneous tone of the trachea. Pretreatment with PDA (0.1-10 microM) converted relaxant responses to high concentrations of the methylxanthines into contractions. PDA produced no equivalent effect against isoprenaline. Pretreatment with verapamil (1 or 10 microM), nifedipine (0.1 microM) or incubation with Ca(2+)-free, EGTA (0.1 mM)-containing physiological salt solution (PSS) suppressed the contraction produced by caffeine or theophylline in PDA (5 microM)-treated tissues. 3. The ability of PDA (5 microM) to convert caffeine-induced relaxation into caffeine-induced contraction was retained in tissues pretreated with a combination of atropine (1 microM) and mepyramine (1 microM) and in tissues denuded of the airway epithelium. 4. Caffeine (10 microM-5 mM), theophylline (10 microM-5 mM) and isoprenaline (1 nM-1 microM), each relaxed trachea contracted with histamine (0.1 mM). The relaxation induced by caffeine, theophylline and isoprenaline was markedly reduced in the presence of PDA (5 microM) and the responses to high concentrations of caffeine and theophylline, but not those to isoprenaline, were reversed to contractions. Verapamil (10 microM) prevented the effects of PDA against caffeine- or theophylline-induced relaxation. 5. PDA (1 microM) enhanced the tracheal spasm produced by caffeine (10 mM) and theophylline (10 mM) in indomethacin (2.8 microM)-treated trachea maintained at 20 degrees C. This enhancement was reduced in the presence of verapamil (10 microM). 6. Tested in trachea bathed by K+-rich (40 mM), Ca2+-free PSS, CaCl2 (0.1-20 mM) caused concentration-dependent spasm. PDA (1-5 MicroM) did not significantly modify the shape or position of the log concentration-effect curve for CaCl2. In contrast, verapamil (1 and 10 MicroM) antagonized CaCl2.7. Tested in trachea bathed by indomethacin (2.8 MicroM)-containing PSS, ryanodine (1-100 MicroM) caused concentration-dependent spasm. PDA (5 MicroM) potentiated ryanodine. Verapamil (10 MicroM) inhibited ryanodine in inducing spasm and suppressed the ability of PDA to potentiate ryanodine.8. It is concluded that, in guinea-pig isolated trachea, PDA augments the spasmogenic activity of the methylxanthines and ryanodine. This effect of PDA does not result from PDA-induced suppression of spontaneous tone, from increased cellular entry of Ca2+ through L-type channels or from sensitization of the intracellular contractile machinery to activator Ca2+. The evidence suggests, instead, that PDA facilitates methylxanthine- or ryanodine-induced release of Ca2+ from the intracellular store.

The individual airway responsiveness to inhaled, nebulized methacholine (MeCh) was estimated in normal volunteers, measuring specific airway conductance (sGAW). The dose of MeCh was increased logarithmically until a 60-65% reduction from baseline sGAW or an asymptotic approach to a maximal response was attained. The concentration of MeCh that caused a 35% reduction in sGAW (PC35), the dose that caused a 62.5% reduction in sGAW, the slope of the straight, central part of the log-dose-response curve (LDRC), the slope of the straight, initial part of the dose-response curve, the maximal response attainable (Emax) and the dose causing a half-maximal response (ED50) were derived. These parameters were transformed as necessary to attain normality of distribution. Relationships between them were examined by measuring the correlations between their transformed values. The ED50 was taken to represent the least biased estimate of the sensitivity to MeCh. The PC35 was the best practical estimate of sensitivity. The Emax was taken to represent the least biased estimate of the reactivity to MeCh. The slope of the LDRC was the best practical estimate of reactivity. The sensitivity and reactivity varied independently in these normal subjects. Each was also independent of the baseline sGAW.

1. Studies of mechanical activity and 86Rb+ efflux have been made in bovine isolated trachealis with the objectives of: (a) identifying which of the beta-adrenoceptor subtypes mediates the opening of plasmalemmal K(+)-channels, (b) gaining further insight into the properties of the novel, long-acting beta 2-adrenoceptor agonist, salmeterol and (c) clarifying the role of K(+)-channel opening in mediating the mechano-inhibitory actions of agonists at beta-adrenoceptors. 2. In bovine trachealis muscle strips precontracted with histamine (460 microM), isoprenaline (0.1 nM-1 microM), procaterol (0.1-10 nM) and salmeterol (0.1-10 nM) each caused concentration-dependent relaxation. 3. ICI 118551 (10 nM-1 microM) antagonized isoprenaline, procaterol and salmeterol in suppressing histamine-induced tone of the isolated trachealis muscle. The antagonism was concentration-dependent. In contrast, CGP 20712A (10 nM-1 microM) failed to antagonize isoprenaline, procaterol or salmeterol. 4. Salmeterol (1-10 microM) antagonized isoprenaline in relaxing strips of bovine trachea which had been precontracted with carbachol (1 microM). 5. Cromakalim (10 microM), isoprenaline (100 nM-10 microM), procaterol (10 nM-1 microM) and salbutamol (100 nM-10 microM) each promoted the efflux of 86Rb+ from strips of bovine trachealis muscle preloaded with the radiotracer. In contrast, salmeterol (100 nM-10 microM) failed to promote 86Rb+ efflux. 6. CGP 201712A (1 microM), ICI 118551 (100 nM) and salmeterol (1 microM) did not themselves modify 86Rb+ efflux from trachealis muscle strips, nor did they affect the promotion of 86Rb+ efflux induced by cromakalim (10 microM). In contrast, CGP 20712A (1 microM) and ICI 118551 (100nM) were each able to inhibit the promotion of 86Rb+ efflux induced by isoprenaline (1 microM) or procaterol (100 nM). Furthermore,salmeterol (10 microM) inhibited isoprenaline (1 microM)-induced promotion of 86Rb+ efflux.7. It is concluded that, in bovine trachealis, activation of either beta l- or beta 2-adrenoceptors can promote the opening of 86Rb+-permeable K+-channels in the plasmalemma. The failure of salmeterol to promote plasmalemmal K+-channel opening may reflect, not its selectivity in activating beta 2- as opposed to beta 1-adrenoceptors, but rather its low intrinsic efficacy at beta 2-adrenoceptors. The opening of plasmalemmal K+-channels plays a supportive rather than a crucial role in mediating the mechano-inhibitory effects of agonists at beta-adrenoceptors acting on trachealis muscle.

1. Mechanical and electrophysiological studies of guinea-pig isolated trachealis have been made with the objectives of: (a) identifying which of the beta-adrenoceptor subtypes mediates the opening of plasmalemmal K(+)-channels, (b) gaining further insight into the properties of the novel, long-acting beta-adrenoceptor agonist, salmeterol and (c) clarifying the role of K(+)-channel opening in mediating the relaxant actions of agonists at beta-adrenoceptors. 2. Noradrenaline (10 nM-100 microM) caused a concentration-dependent increase in the rate of beating of guinea-pig isolated atria. The selective beta 1-adrenoceptor blocking drug, CGP 20712A (100 nM-10 microM) caused concentration-dependent antagonism of noradrenaline. The selective beta 2-adrenoceptor blocking drug, ICI 118551, also produced concentration-dependent antagonism of noradrenaline, but only when used in concentrations greater than 300 nM. 3. Cromakalim (100 nM-10 microM), isoprenaline (1-100 nM), procaterol (0.1-30 nM), salbutamol (1 nM-1 microM), salmeterol (1-100 nM) and theophylline (1 microM-1 mM) each caused concentration-dependent suppression of the spontaneous tone of guinea-pig isolated trachealis. 4. ICI 118551 (10 nM-1 microM) antagonized isoprenaline, procaterol and salmeterol in suppressing the spontaneous tone of the isolated trachea. The antagonism was concentration-dependent. In contrast, ICI 118551 (1 microM) antagonized neither cromakalim nor theophylline. CGP 20712A (up to 1 microM) failed to antagonize cromakalim, isoprenaline, procaterol, salmeterol or theophylline. In trachea treated with indomethacin (2.8 microM) and carbachol (10 microM), salmeterol (1 microM) antagonized the effects of isoprenaline but not aminophylline. 5. Intracellular electrophysiological recording from guinea-pig isolated trachealis showed that the relaxant effects of cromakalim (10 microM), isoprenaline (100 nM), procaterol (10 nM) and salbutamol(10 nM- 1 microM) were accompanied by the suppression of spontaneous electrical slow waves and by cellular hyperpolarization. In contrast, the relaxant effects of salmeterol (10 nM- 1 microM) were not accompanied by significant cellular hyperpolarization.6. CGP 20712A (1 microM) inhibited the hyperpolarization but not the relaxation induced by isoprenaline(100 nM). In contrast ICI 118551 (100 nM) inhibited both the hyperpolarization and the relaxation induced by isoprenaline (100 nM). Neither CGP 20712A (1 microM) nor ICI 118551 (100 nM) inhibited the hyperpolarization induced by cromakalim (10 microM). Salmeterol (1 microM) inhibited the hyperpolarization induced by isoprenaline (100 nM) but not that induced by cromakalim (10 microM).7. It is concluded that activation of either beta l- or beta 2-adrenoceptors can promote the opening of K+-channels in the trachealis plasmalemma. The poor ability of salmeterol to hyperpolarize trachealis muscle reflects neither its selectivity in activating beta 2-adrenoceptors as opposed to beta 1-adrenoceptors nor a non-specific action in stabilizing the cell membrane. Instead, it may reflect low intrinsic efficacy of the drug at beta 2-adrenoceptors. The opening of plasmalemmal K+-channels plays a supportive rather than a crucial role in mediating the tracheal relaxant actions of agonists at beta-adrenoceptors.

Freshly-dispersed bovine trachealis cells were used for recording by the patch clamp technique of whole-cell and unitary currents through Ca(2+)-channels. Whole-cell Ca(2+)-current (ICa) activated at -40 mV and appeared to be carried by a single type of Ca(2+)-channel. Inactivation of ICa was increased by increasing the concentration of free Ca2+ within the recording pipette but reduced by using Ba2- as the charge carrier. Steady-state inactivation studies showed that the Ca(2+)-channels were half-maximally available following a conditioning depolarization to -35 mV. A two-pulse protocol showed that ICa induced by the step to a test potential was inversely related to ICa induced by the step to the conditioning potential. Unitary Ba(2+)-currents were activated at a threshold of -30 mV and had a reversal potential of +41 mV. The channel carrying the unitary Ba(2+)-currents had a slope conductance of 23 pS. Steady-state inactivation studies showed that the unitary Ba(2+)-currents were half-maximally available at a holding potential of -28 mV. ICa and unitary Ba(2+)-currents were inhibited by nifedipine (10 nM-1 microM) but augmented by Bay K 8644 (10 microM). It is concluded that the plasmalemma of bovine trachealis muscle contains a single population of voltage-dependent Ca(2+)-channels of the L-type. These channels may be subject to inactivation primarily by an increase in the concentration of free Ca2+ on the cytosolic side of the plasmalemma and secondarily by a voltage-dependent mechanism. Overlap of the inactivation and activation curves of ICa may allow the passage of 'window current' through the Ca(2+)-channels during sustained depolarization.

An approximately steady-state reduction of specific airway conductance was induced in healthy human subjects by means of an individualized inhaled methacholine loading dose followed by a maintenance dose regime. Tested against this background bronchoconstriction, the xanthine analogue SDZ MKS 492, when administered as a single oral dose of 40 mg, showed a significant bronchodilator action, which lasted for up to 5.5 h. Bronchodilatation was not seen after administration of 10 or 20 mg doses. SDZ MKS 492 inhaled as a dry powder had a bronchodilator action that was small, most evident with the 12 mg dose and transient. The peak relief of imposed bronchoconstriction was 29% and the apparent half-time of removal of SDZ MKS 492 from its site of action was 5-6 min. Inhaled SDZ 492 had a bitter taste that was not masked by inclusion of menthol and aspartame in the formulation. The bronchodilatation seen in laboratory animals can also be produced by SDZ MKS 492 in man when administered orally or by inhalation. Its magnitude correlates better with the plasma concentration of parent drug than with that of either of the identified metabolites. Dispositional processes in the lung abbreviate its action after administration by inhalation.

1. The adrenergic neurone blocking agents, guanethidine and bretylium, have been tested for inhibitory activity against the actions of some relaxant drugs (BRL 38227, noradrenaline, sodium nitroprusside, theophylline) in vascular, intestinal and uterine smooth muscle. 2. In guinea-pig isolated taenia caeci pre-contracted with KCl (25 mM), BRL 38227 (0.1-10 microM) and noradrenaline (10 nM-100 microM) each caused concentration-dependent relaxation. Guanethidine and bretylium (50 microM) each antagonized the relaxation to BRL 38227 but not that to noradrenaline. At high concentration (500 microM), the adrenergic neurone blocking agents antagonized the action of BRL 38227 and, to some extent, that of noradrenaline. 3. In rat isolated aorta pre-contracted with noradrenaline (300 nM), BRL 38227 (0.0125-3.2 microM) and sodium nitroprusside (0.3-100 nM) each produced concentration-dependent smooth muscle relaxation. Guanethidine and bretylium (5-500 microM) each antagonized the action of BRL 38227 without antagonizing that of sodium nitroprusside. 4. Rats were pretreated with 17-beta oestradiol benzoate. Tension waves were then induced from segments of isolated, oestrogen-dominated uterus by transmural electrical stimulation or by oxytocin (0.2 nM). These tension waves were inhibited by BRL 38227 (0.025-3.2 microM) or theophylline (0.05-0.8 mM) in a concentration-dependent manner. Guanethidine (50 microM) antagonized the action of BRL 38227 in both the electrically- and oxytocin-driven tissues. In the electrically-driven tissues, guanethidine (50 microM) did not antagonize the inhibition to theophylline. 5. In KCl (25 mM)-treated guinea-pig taenia caeci, guanethidine (50 microM) inhibited the efflux of 86Rb+ evoked by BRL 38227 (10 microM) but not that evoked by noradrenaline (10 microM). In contrast, apamin(100 nM) reduced the efflux of 86Rb+ which was promoted by noradrenaline, but did not affect efflux induced by BRL 38227.6. It is concluded that the adrenergic neurone blocking agents, guanethidine and bretylium (each at 50 microM), selectively inhibit the relaxant action of BRL 38227 in vascular, intestinal and uterine smooth muscle. If this inhibition reflects direct blockade of the K+-channel (KKCO) which is opened by BRL 38227, then the adrenergic neurone blocking agents act as inhibitors selective for KKCO as opposed to the small, apamin-sensitive (SKCa) and large (BKca) conductance, Ca2"-dependent K+-channels.

SDZ PCO 400 (30 nM-100 microM) suppressed the spontaneous tone of guinea-pig isolated trachealis. Glibenclamide (1-10 microM), phentolamine (100 microM), guanethidine (50 microM) and bretylium (50 microM) each antagonized SDZ PCO 400 without antagonizing isoprenaline or theophylline. Charybdotoxin (100 nM) failed to antagonize SDZ PCO 400 but antagonized theophylline. The relaxant action of SDZ PCO 400 was ablated when spasm was induced by a K(+)-rich (120 mM) medium. In bovine and guinea-pig trachea, SDZ PCO 400 (10 microM) suppressed spasm evoked by lower (less than 40 mM) but not higher (greater than 40 mM) concentrations of KCl. In guinea-pig trachea the relaxant action of SDZ PCO 400 was associated with suppression of electrical slow waves and with marked cellular hyperpolarisation. SDZ PCO 400 (0.5 and 10 microM) promoted the efflux of 86Rb+ from bovine trachealis, an effect inhibited by glibenclamide (1 microM). It is concluded that the tracheal relaxant action of SDZ PCO 400 is associated with the opening of a plasmalemmal K(+)-channel analogous to the ATP-sensitive K(+)-channel observed in insulin-secreting cells.

1. We have studied the ability of some adrenergic neurone blocking agents to inhibit the tracheal relaxant actions of isoprenaline, theophylline and the potassium channel openers (KCOs) BRL 38227, pinacidil and RP 52891. 2. BRL 38227, isoprenaline, pinacidil, RP 52891 and theophylline each caused concentration-dependent suppression of the spontaneous tone of guinea-pig isolated trachealis. The maximal relaxant effects of isoprenaline and pinacidil were equal to that of theophylline. In contrast, the maximal effects of BRL 38227 and RP 52891 were approximately 85-95% of that of theophylline. 3. Guanethidine (5-500 microM) did not itself modify the spontaneous tone of the trachealis muscle but antagonized BRL 38227 in a concentration-dependent manner. Guanethidine (50 microM) also antagonized pinacidil and RP 52891. However, guanethidine did not antagonize either isoprenaline or theophylline. 4. Bretylium (50 microM) did not itself modify the spontaneous tone of the trachealis muscle but antagonized BRL 38227, pinacidil and RP 52891. Bretylium did not antagonize either isoprenaline or theophylline. 5. Guanidine (50 and 500 microM) did not itself modify the spontaneous tone of the trachea and failed to modify the tracheal relaxant activity both of BRL 38227 and theophylline. 6. BRL 38227 (1 and 10 microM) stimulated, in a concentration-dependent manner, the efflux of 86Rb+ from strips of bovine trachealis muscle that had been pre-loaded with the radiotracer. Guanethidine (50 microM), bretylium (50 microM) and debrisoquine (50 microM) did not themselves modify the efflux of 86Rb+ from bovine trachealis but each of these agents markedly inhibited the stimulant effect of BRL 38227 (10 microM) on 86Rb+ efflux.7. It is concluded that the adrenergic neurone blocking agents guanethidine and bretylium can inhibit the tracheal relaxant actions of KCOs such as BRL 38227, pinacidil and RP 52891 without antagonizing isoprenaline or theophylline. The ability of the adrenergic neurone blocking agents to antagonize BRL 38227 in promoting 86Rb+ efflux from trachealis muscle may suggest that the adrenergic neurone blocking agents act to prevent the opening of the plasmalemmal K+-channel that is involved in the tracheal relaxant actions of the KCOs.

1. Potassium channel opening drugs (KCOs) include benzopyrans such as cromakalim, cyanoguanidines such as pinacidil and tetrahydrothiopyrans such as RP 49356. 2. While clinical trials have indicated that cromakalim may be of benefit in the treatment of nocturnal asthma, it remains to be determined whether KCOs will find a place in our armamentarium of clinically useful anti-asthma agents. 3. KCOs inhibit the spontaneous tone of airways smooth muscle in vitro, an action associated with membrane hyperpolarization towards the potassium equilibrium potential and with the promotion of 86Rb+ or 42K+ efflux from the muscle cells. KCOs suppress spasm of airways smooth muscle evoked by low (< 40 mM) but not high (> 40 mM) concentrations of KCl. Their relaxant effects in airways smooth muscle can be attenuated by a variety of agents (including sulphonylureas) known to inhibit the opening of plasmalemmal K(+)-channels. 4. The KCOs open an ATP-sensitive K(+)-channel (KATP) in the plasmalemma. KATP is not open under normal circumstances and does not play an important role in determining the strong outward rectifying behavior of the cell membrane. The biochemical mechanisms by which the KCOs promote the opening of KATP remain to be elucidated but probably do not involve channel phosphorylation consequent to the intracellular accumulation of cAMP. 5. By causing hyperpolarization of the plasmalemma, the KCOs inhibit the cellular influx of Ca2+ through voltage-dependent channels. Relaxation follows both as a direct consequence of the fall in cytosolic free Ca2+ and also as a consequence of reduced production of phosphoinositide second messengers. The KCOs may also inhibit Ca2+ uptake by, and hence Ca2+ release from, the sarcoplasmic reticulum. 6. KCOs can inhibit cholinergic and non-adrenergic, non-cholinergic (NANC) excitatory neuroeffector transmission in the airways by glibenclamide-sensitive mechanisms which may involve inhibition of neurotransmitter release. The KCOs do not attenuate NANC inhibitory neuroeffector transmission, suggesting that KATP may not be expressed in neurones of this type. 7. The active enantiomer of cromakalim has been found to be effective in alleviating nocturnal asthma at plasma concentrations just threshold for relaxing human airways smooth muscle in vitro. The clinical efficacy of cromakalim may therefore depend on an action other than the direct relaxation of airways smooth muscle. Animal studies indicate that KCOs can reduce airway hyper-reactivity at sub-bronchodilator doses. The mechanism of this effect remains to be elucidated and may not crucially depend upon inhibition of neurotransmitter release within the lung.

The cromakalim-like KCOs relax airways smooth muscle by an action that is associated with the opening of plasmalemmal K(+)-channels. The K(+)-channel involved may be analogous to the ATP-sensitive K(+)-channel identified in pancreatic beta-cells. It is unlikely to be open under normal circumstances and plays little role in determining the strong outward rectifying behaviour of the plasmalemma of the airways smooth muscle cell. K(+)-channel opening may cause relaxation of the airways smooth muscle cell by mechanisms additional to inhibition of Ca2+ influx through L-type VOCs. The cromakalim-like KCOs have bronchodilator activity in vivo and can depress NANC excitatory neuroeffector transmission in the lung at concentrations smaller than those required to relax airways smooth muscle. The mechanism of action of cromakalim in alleviating nocturnal asthma may not involve direct relaxation of airways smooth muscle. It is possible that cromakalim may instead act to inhibit the mechanisms underlying airway hyper-reactivity.

1. The ability of cromakalim to modulate several different types of neuroeffector transmission has been assessed in guinea-pig isolated trachea. 2. In trachea treated with propranolol (10(-6) M) and indomethacin (2.8 x 10(-6) M), stimulation of the extrinsic vagal nerves evoked contractions which were blocked by hexamethonium (5 x 10(-4) M) or by tetrodotoxin (TTX; 10(-6) M). Cromakalim (10(-5) M) caused a two fold rightward shift of the frequency-response curve. 3. In carinal trachea treated with propranolol and indomethacin, transmural stimulation evoked an initial, rapid contraction followed by a more sustained secondary contraction. The initial, rapid contractile response was virtually ablated by atropine (10(-6) M) or by TTX but was resistant to hexamethonium. Cromakalim (10(-8)-10(-5) M) caused a concentration-dependent rightward shift of the frequency-response curve for the initial contraction. 4. In carinal trachea treated with atropine, propranolol and indomethacin, transmural stimulation evoked only the secondary (non-adrenergic, non-cholinergic (NANC] contractile responses. These were markedly reduced by TTX but were resistant to hexamethonium. Cromakalim (10(-8)-10(-5) M) suppressed the NANC contractile responses in a concentration-dependent manner. This action could be offset by glibenclamide (10(-6) M). 5. In trachea treated with atropine, histamine (10(-4) M), propranolol and indomethacin, transmural stimulation evoked NANC relaxant responses. Cromakalim (up to 10(-5) M) was without effect on the frequency-response curve for the stimulation of NANC inhibitory nerves. 6. Tested on trachea bathed by drug-free Krebs solution, cromakalim (10(-7)-10(-5) M) caused concentration-dependent suppression of tracheal tone. In trachea treated with propranolol and indomethacin, cromakalim (10- 7-1O- 5 M) caused concentration-dependent antagonism of acetylcholine (ACh). In trachea treated with atropine, propranolol and indomethacin, cromakalim (up to 10- 5M) failed to antagonize effects of either histamine or substance P.7. It is concluded that cromakalim can inhibit cholinergic (excitatory) neuroeffector transmission in the trachea but only at a concentration having demonstrable inhibitory activity against the action of exogenous ACh and the spontaneous tone of the airways smooth muscle. In contrast, cromakalim may depress NANC excitatory (putative peptidergic) neuroeffector transmission at a concentration below that exerting inhibitory activity on airways smooth muscle. Cromakalim does not concurrently depress NANC inhibitory neuroeffector transmission. Depression of NANC excitatory neuroeffector transmission could explain the ability of cromakalim to suppress airway hyperreactivity or bronchial asthma at doses lacking direct relaxant effect on airways smooth muscle.

1. A study has been made, in guinea-pig isolated trachealis, of the effects of charybdotoxin in modulating (a) the activity of large conductance K(+)-channels, (b) the spontaneous electrical activity of intact cells and (c) the mechanical effects of some bronchodilator drugs. 2. Single smooth muscle cells were isolated from guinea-pig trachealis by enzymic digestion and were studied by the patch clamp recording technique. Recordings were made from outside-out plasmalemmal patches when the medium bathing the external surface of the patches contained 1.2 mM Ca2+ and 6 mM K+ while that bathing the cytosolic surface contained 0.1 microM Ca2+ and 140 mM K+. Charybdotoxin (100 nM), applied to the external surface of patches held at 0 mV, abolished the unitary currents associated with the opening of large conductance K(+)-channels. 3. Opened segments of guinea-pig trachea were used for the simultaneous recording of membrane potential and tension changes. In these experiments charybdotoxin (100 nM) caused the conversion of spontaneous electrical slow waves into spike-like action potentials. This effect was accompanied by a very small reduction in resting membrane potential. 4. Tissue bath recording showed that charybdotoxin (100 nM) increased the spontaneous mechanical tone of the tissue, antagonized (2.8 fold in each case) the relaxant actions of isoprenaline and theophylline but did not antagonize the relaxant actions of cromakalim or RP 49356. 5. It is concluded that charybdotoxin is an effective inhibitor of large conductance K(+)-channels in guinea-pig trachealis cells. The ability of charybdotoxin to convert spontaneous slow waves into spike-like action potentials suggests that the large, charybdotoxin-sensitive, K+-channels play an important role in determining the strong outward rectifying behaviour of the cells. The ability of charybdotoxin to antagonize isoprenaline and theophylline, but not to antagonize cromakalim and RP 49356, suggests that opening of the large conductance, charybdotoxin-sensitive K+-channel is implicated in the action of the former but not the latter pair of bronchodilator drugs.

1. Single smooth muscle cells were isolated from bovine trachealis by enzymic digestion. The properties of large conductance plasmalemmal K(+)-channels in these cells were studied by the patch-clamp recording technique. 2. Recordings were made from inside-out plasmalemmal patches when [K+] was symmetrically high (140 mM) and when [Ca2+] on the cytosolic side of the patch was varied from nominally zero to 10 microM. Large unitary currents of both Ca(2+)-dependent and -independent types were observed. Measured between + 20 and + 40 mV, the slope conductances of the channels carrying these currents were 249 +/- 18 pS and 268 +/- 14 pS respectively. 3. Lowering [K+] on the cytosolic side of the patches from 140 to 6 mM, shifted the reversal potentials of the two types of unitary current from approximately zero to much greater than + 40 mV, suggesting that both currents were carried by K(+)-channels. 4. The Ca(2+)-dependent and -independent K(+)-channels detected in inside-out plasmalemmal patches could also be distinguished on the basis of their sensitivity to inhibitors (tetraethylammonium (TEA), 1-10 mM; Cs+, 10 mM; Ba2+, 1-10 mM; quinidine, 100 microM) applied to the cytosolic surface of the patches. 5. Recordings were made from outside-out plasmalemmal patches when [K+] was symmetrically high (140 mM) and when [Ca2+] on the cytosolic side of the patch was varied from nominally zero to 1 microM. Ca(2+)-dependent unitary currents were observed and the slope conductance of the channel carrying these currents was 229 +/- 5 pS. 6. Activity of the Ca2+-dependent K+-channel detected in outside-out patches could be inhibited by application of TEA (1 mM), Cs+ (10mM), Ba2(+210mM) or quinidine (100 microM) to the external surface of the patch. 4-Aminopyridine (4-AP; 1 mM) was ineffective as an inhibitor. 7. The activity of the Ca2+-dependent K+-channel recorded from outside-out patches was reversibly inhibited by charybdotoxin (100 nM). 8. When whole-cell recording was performed, the application of a depolarizing voltage ramp evoked outward current which was dependent on the [Ca2 +] in the recording pipette and which could be reversibly inhibited by charybdotoxin (50 nM-I microM) applied to the external surface of the cell.9. We conclude that bovine trachealis cells are richly endowed with charybdotoxin-sensitive, large conductance, Ca2 +-dependent K+-channels. These channels carry most of the outward current evoked by a depolarizing ramp and could play a major role in determining the outward rectifying properties of the trachealis cells. The role of the large Ca2 + -independent K+ -channels remains unclear.

In Triton X-100-skinned trachealis muscle, neither papaverine nor AH 21-132 modified responses to Ca2+. The (-)-enantiomer of AH 21-132 was more potent than the (+)-enantiomer both in relaxing intact trachealis muscle and in inhibiting tracheal cAMP phosphodiesterase (PDE). AH 21-132 (0.6 microM) potentiated forskolin in causing tracheal relaxation but did not potentiate isoprenaline, cromakalim or sodium nitrate. AH 21-132 (2 microM) potentiated all four agents in relaxing the trachea. AH 21-132 (1 microM) potentiated forskolin in increasing tissue cAMP content and, in higher concentration, itself increased tissue cAMP. Electrical effects of AH 21-132 included suppression of spontaneous slow waves and cellular hyperpolarisation. It is concluded that AH 21-132 lacks a direct depressant effect on the intracellular contractile machinery. The weight of evidence suggests that AH 21-132-induced relaxation results from inhibition of cAMP-PDE. However, in common with other PDE inhibitors. AH 21-132 increases tissue cAMP content only at concentration greater than that required to cause full relaxation.

The benzonaphthyridine derivative, AH 21-132, has non-specific relaxant effects in isolated airways smooth muscle. The action of AH 21-132 in trachealis muscle is not antagonised by propranolol but AH 21-132 is slightly potentiated by epithelium removal. Electrophysiological recording from guinea-pig trachealis shows that AH 21-132-induced relaxation is accompanied by suppression of electrical slow waves and by cellular hyperpolarisation. Unlike theophylline, AH 21-132 does not cause spasm of cooled (12 degrees C), indomethacin-treated trachealis muscle, nor does it act as an antagonist at adenosine A1 receptors. AH 21-132 does not depress the Ca2+ sensitivity or responsiveness of Triton X-100 skinned trachealis fibres. In tracheal relaxant concentrations, AH 21-132 selectively inhibits cAMP phosphodiesterase (PDE) compared with cGMP-PDE. The (-)-enantiomer of AH 21-132 is more potent than its (+)-enantiomer both in causing tracheal relaxation and in inhibiting cAMP-PDE. When tested on PDE isoenzymes separated from bovine trachealis and guinea-pig cardiac ventricles, AH 21-132 exhibits selectivity as an inhibitor of the isoenzyme types III and IV. AH 21-132 increases the trachealis content of cAMP and cGMP, but only in concentration greater than that required fully to suppress the mechanical tone of the tissue. AH 21-132 has bronchodilator activity in anaesthetised, ventilated guinea-pigs when administered intraduodenally, intravenously or by inhalation. Inhaled AH 21-132 also provides bronchodilatation in healthy human volunteers in whom bronchoconstriction has been induced by inhaled methacholine.

The smooth muscle relaxant, AH 21-132, was tested for its inhibitory effect on the cyclic nucleotide phosphodiesterase (PDE) activities fractionated from guinea-pig cardiac ventricle and bovine trachealis muscle. Both tissues yielded significant PDE-I and PDE-II activities. The cardiac ventricle also contained a significant amount of PDE-III whilst the trachealis contained PDE-IV. AH 21-132 inhibited PDE-III and PDE-IV selectively (Ki values 0.30-0.55 microM) compared with PDE-I and PDE-II (Ki values 20-140 microM).

Experiments have been performed using guinea-pig and bovine trachealis in order to determine whether cromakalim and RP 49356 share the same relaxant action and to analyse the mechanisms underlying this action. RP 49356 was approximately 3 times less potent than cromakalim in suppressing the spontaneous tone of guinea-pig trachea and, like cromakalim, was antagonised by glibenclamide and by phentolamine. Biochemical studies showed that relaxant concentrations of cromakalim and RP 49356 did not alter the cAMP or cGMP content of guinea-pig trachealis muscle and did not inhibit cAMP or cGMP hydrolysis by tracheal homogenates. Like cromakalim, RP 49356 caused marked hyperpolarisation of guinea-pig trachealis cells. Patch clamp recording using inside-out membrane patches from bovine trachealis showed that cromakalim, RP 49356, glibenclamide and phentolamine were each without effect on the open state probability (Popen) of large conductance, Ca(2+)-activated K(+)-channels. We conclude that cromakalim and RP 49356 share a similar action in opening K(+)-channels in the trachealis cell membrane. This action probably does not involve the intracellular accumulation of cyclic nucleotides and the channel involved is not the large conductance, Ca(2+)-dependent K(+)-channel.

1. Isolated segments of guinea-pig trachea or perfused tracheal tubes were arranged for the recording of trachealis tension changes in Krebs solution containing indomethacin (2.8 microM). 2. In opened tracheal segments, epithelium removal caused modest (2-3 fold) potentiation of the effects of acetylcholine (ACh) and methacholine (MeCh) but failed to potentiate carbachol (CCh), bethanechol (BeCh), oxotremorine or KCl. 3. Pretreatment with ecothiopate potentiated effects of ACh and MeCh but not of CCh or BeCh. Removal of epithelium in ecothiopate-treated tissue potentiated effects of ACh and MeCh but not of CCh or BeCh. 4. Guinea-pig ileum challenged with ACh was used as a bioassay system for cholinesterase activity. Scrapings of tracheal epithelium did not hydrolyse ACh. 5. Histochemical staining revealed no fibres positive for acetylcholinesterase or pseudocholinesterase in the tracheal epithelium. However, the underlying tissues contained acetylcholinesterase-positive nerve fibres and the trachealis muscle itself stained positively for pseudocholinesterase activity. 6. Neither tetrodotoxin (3 microM) nor hexamethonium (500 microM) modified the ability of epithelium removal to potentiate ACh. 7. In perfused tracheal tubes where spasmogens were added to the luminal perfusate, epithelium removal potentiated effects of ACh (31 fold), CCh (10 fold), oxotremorine (2 fold) and KCl. 8. In perfused tracheal tubes where spasmogens were added to the Krebs solution superfusing the adventitial surface of the tissue, epithelium removal significantly reduced the potency of CCh, oxotremorine and KCl. 9. It is concluded that the selectivity and magnitude of the potentiation of cholinomimetics caused by epithelium removal depends on the route by which the cholinomimetic agent gains access to the trachealis muscle. The potentiation of acetic acid esters of choline seen in opened tracheal segments does not reflect the loss of epithelial cholinesterase activity and does not depend on the activity of nervous reflex arcs in the tracheal wall. The reduced potency of adventitially-applied cholinomimetics and KCl seen in epithelium-denuded tissue strongly suggests that the epithelium can moderate trachealis sensitivity to cholinomimetic agents not only by releasing epithelium-derived relaxing factor but also by acting as a barrier to drug diffusion.

In guinea-pig trachealis, depolarized by a K(+)-rich medium, Ca2+ (0.01-10 mM) caused concentration-related spasm. Verapamil (0.5-5 microM), cinnarizine (10-100 microM) and trifluoperazine (16-160 microM) each produced concentration-dependent antagonism of Ca2+ characterized by a rightward and downward displacement of the log concentration-effect curve for Ca2+. The rank order of potencies of these antagonists, measured as the IC75 against Ca2+ (10 mM)-induced contraction of depolarized trachea, was verapamil (5.6 microM) greater than cinnarizine (59 microM) greater than trifluoperazine (91 microM). In skinned trachea, verapamil in concentrations up to 100 microM did not modify the concentration-effect curve for Ca2+. In contrast, cinnarizine (59-177 microM) diminished the sensitivity and trifluoperazine (273 microM) decreased the responsiveness of the tissue to Ca2+. In skinned trachea, trifluoperazine (91 microM) produced greater inhibition of Ca2+ (10 microM)-induced contraction after 120 min than after 30 min of incubation. Verapamil (100 microM) and cinnarizine (177 microM) were devoid of inhibitory effect against the 10 microM Ca2+ standard. In skinned trachea, changes in the Ca2+ concentration-effect curve produced by cinnarizine (177 microM) were reversed after washout whilst those induced by trifluoperazine (273 microM) persisted. It is concluded that distinct differences exist between the three calcium antagonists examined. The action of verapamil is restricted to the plasmalemma. That of cinnarizine and trifluoperazine is exerted both on the plasma membrane and upon the intracellular contractile machinery.

1. AH 21-132 is being investigated as a potential chemotherapeutic agent for bronchial asthma. The present experiments were designed to determine whether AH 21-132 shares the activity of theophylline as an antagonist at adenosine A1 receptors and to assess its potency as a relaxant in intestinal smooth muscle. 2. In the transmurally-stimulated guinea-pig ileum, theophylline (1 mM), but not AH 21-132 (1 and 10 microM), antagonized twitch depression induced by adenosine. Higher concentrations (100 microM and 1 mM) of AH 21-132 themselves had a depressant effect. Neither theophylline (1 mM) nor AH 21-132 (1 and 10 microM) antagonized twitch depression induced by noradrenaline. 3. AH 21-132 (100 microM and 1 mM) depressed maximum contractions of ileum induced by both acetylcholine (ACh) and histamine. 4. In ileum treated with hyoscine (1 microM), AH 21-132 (greater than 10 microM) caused a concentration-dependent depression of the log concentration-effect curve for potassium chloride. 5. Simultaneous extracellular electrophysiological and mechanical recording from taenia caeci showed that AH 21-132 (100 microM-1 mM) inhibited spontaneous tension waves and their associated bursts of electrical spike activity. 6. Intracellular electrophysiological recording from taenia caeci showed that the mechano-inhibitory effect of 1 mM AH 21-132 was accompanied by abolition of spontaneous spike activity. Following spike abolition, the membrane potential assumed a value very close to that observed during periods of electrical quiescence prior to drug exposure. 7. AH 21-132 inhibited the activity of cyclic AMP-dependent and cyclic GMP-dependent phosphodiesterases derived from homogenates of ileal smooth muscle. The effective concentration ranges were 0.1-1OOO microM and 1-1000 microM, respectively. Theophylline, too, inhibited these enzymes but in each case was less potent than AH 21-132. 8. It is concluded that AH 21-132 is devoid of antagonist activity at adenosine Al receptors which modulate ACh release from intramural cholinergic nerves in the ileum. At concentrations greater than IO microM, AH 21-132 has a relaxant effect on intestinal smooth muscle characterized by suppression of spontaneous action potentials but by minor change in resting membrane potential. AH 21-132 previously has been reported to depress the spontaneous tone of trachealis muscle with an EC50 value of less than lO microM and the present experiments therefore show that this agent is much less potent in inhibiting intestinal muscle. This potency difference cannot be attributed to a tissuerelated difference in the potency of AH 21-132 as an inhibitor of cyclic AMP- or cyclic GMPdependent phosphodiesterases.

Aminophylline (1-1000 mumol l-1) suppressed the spontaneous tone of guinea-pig isolated trachealis in a concentration-dependent manner. In Krebs solution containing acetylcholine (1 mmol l-1), histamine (200 mumol l-1) or K+ (120 mmol l-1) isolated trachealis muscle developed near-maximal tension. The log concentration-effect curve for aminophylline was shifted 20 fold, 3 fold and 4 fold to the right, respectively, in the presence of these spasmogens. Three K+-channel inhibitors were tested: tetraethylammonium (TEA, 8 mmol l-1) did not modify the action of aminophylline, procaine (5 mmol l-1) shifted the log concentration-effect curve for aminophylline 2 fold to the left and 4-aminopyridine (5 mmol l-1) shifted the curve 2.5 fold to the right. Intracellular electrophysiological recording showed that aminophylline 10 mumol l-1 could cause relaxation in the absence of electrical changes. Higher concentrations of aminophylline (100-1000 mumol l-1) suppressed spontaneous slow waves and hyperpolarized the trachealis cells. In the presence of procaine (5 mmol l-1) or TEA (8 mmol l-1), the hyperpolarization induced by aminophylline (1000 mumol l-1) was significantly reduced but its relaxant effect was unchanged. In trachealis skinned of its plasma membranes, tension development induced by Ca2+ (20 mumol l-1) was unaffected either by aminophylline (1000 mumol l-1) or by isoprenaline (1 mumol l-1). In studies of the efflux of 86Rb+ from muscle-rich strips of trachea, aminophylline (100-1000 mumol l-1) was without effect whereas nicorandil (100 and 1000 mumol l-1) increased the efflux rate constant. It is concluded that aminophylline does not directly reduce the sensitivity of the contractile proteins to cytosolic Ca2+. In low concentration (1-10 mumol l-1) its relaxant action is not accompanied by membrane potential change but towards the upper end of its effective concentration range, aminophylline evokes hyperpolarization. This hyperpolarization may involve the opening of K+-channels which are inhibited by procaine and (to a lesser extent) by TEA. These K+-channels may be impermeable to 86Rb+.

1. Intracellular microelectrodes have been used to record the electrical activity of smooth muscle cells of the circular layer from full length strips of mouse colon in vitro. The membrane potential was unstable and showed slow depolarizations (mean amplitude, 10.9 mV; mean frequency, 0.008 Hz; mean duration, 56.4 s). 2. A variable number (mean fifty-six) of rapid oscillations in membrane potential (mean amplitude, 10.2 mV) with a frequency of approximately 2 Hz and a duration of approximately 400 ms were superimposed on each slow depolarization. Occasionally, action potentials arose from the rapid oscillations. The action potentials, but neither the slow depolarizations nor the rapid oscillations, were abolished by 1.0 microM-nifedipine. 3. The majority of the slow depolarizations and the associated rapid oscillations migrated aborally along the colon at a velocity of between 0.5 and 1.5 mm s-1; in the distal colon the slow depolarization was often preceded by a small hyperpolarization. 4. During the rising and plateau phase of the slow depolarization the amplitude of electronic potentials was decreased. Hyperpolarization induced by passing current during the slow depolarization increased the amplitude of the rapid oscillations. 5. Transmural electrical stimulation (single pulses) in the presence of nifedipine evoked (1 mm anal to the stimulating electrodes) an inhibitory junction potential which was sometimes preceded by an excitatory junction potential. The amplitude, of the evoked inhibitory junction potential was decreased during the rising and plateau phase of the slow depolarization. 6. The slow depolarization and the rapid oscillations were abolished by hexamethonium (500 microM), morphine (1-10 microM) and tetrodotoxin (3.1 microM). Atropine (3.5 microM) abolished the rapid oscillations and reduced the amplitude of the slow depolarization. 7. Atropine (3.5 microM) and morphine (10 microM) abolished the evoked excitatory junction potential whilst tetrodotoxin (3.1 microM) abolished both the excitatory and the inhibitory junction potential. 8. It is suggested that the migrating depolarization and accompanying oscillations, which are neurogenic in origin, represent the electrical correlate in the circular muscle layer of the migrating colonic motor complex which has been associated with the propulsion of faecal pellets along the colon.

In guinea-pig isolated trachealis isoprenaline (0.001-0.1 mumol l-1) caused concentration-dependent relaxation. Propranolol (1 mumol l-1) antagonized the effects of isoprenaline by more than 100 fold but did not modify the relaxant action of sodium nitrite. The tracheal relaxant actions of isoprenaline and ATP were unaffected by apamin (0.1 mumol l-1) but apamin profoundly antagonized the effects of noradrenaline and ATP on guinea-pig isolated taenia caeci. Tetraethylammonium (TEA; 8 mmol l-1) and procaine (5 mmol l-1) each evoked tracheal spasm but neither agent antagonized the isoprenaline-evoked relaxation of the trachealis. Trachealis exposed to K+-rich (120 mmol l-1) Krebs solution developed near-maximal tension. Both isoprenaline and sodium nitrite relaxed the K+-depolarized tissue though concentration-effect curves for both relaxants were moved to the right compared to those obtained in non-depolarized tissues. The maximal effect of sodium nitrite was markedly reduced. Intracellular electrophysiological recording showed that isoprenaline (0.01-1 mumol l-1) caused hyperpolarization and reduced or abolished slow wave discharge in trachealis muscle. These effects were accompanied by relaxation. Propranolol (1 mumol l-1) virtually abolished both the electrical and mechanical responses to isoprenaline (0.1 mumol l-1). Apamin (0.1 mumol l-1) did not alter the spontaneous electrical activity of trachealis cells or their electrical and mechanical responses to isoprenaline (0.1 mumol l-1). TEA (8 mmol l-1) caused depolarization and often increased slow wave amplitude and induced spike discharge. Isoprenaline (0.01 mumol l-1) failed to hyperpolarize TEA-treated trachealis cells. Higher concentrations of isoprenaline suppressed TEA-induced spasm, caused hyperpolarization and thereby increased slow wave or spike amplitude. Slow wave or spike frequency decreased as the hyperpolarization progressed but abolition of slow waves or spikes sometimes required more than 4 min exposure to isoprenaline. Procaine (5 mmol l-1) increased the amplitude of slow waves and induced spike discharge. Procaine markedly reduced the hyperpolarization induced by isoprenaline (0.1 and 1 mumol l-1) but had little effect on isoprenaline-induced relaxation. It is concluded that isoprenaline activates beta-adrenoceptors in guinea-pig trachealis and thereby evokes relaxation and hyperpolarization of the smooth muscle. The hyperpolarization does not involve the opening of apamin-sensitive K+-channels and it probably plays a supportive rather than a crucial role in the process by which isoprenaline-induced relaxation is achieved.

Tissue bath experiments showed that potassium chloride (KC1) at 10-40 mmoll-1 evoked spasm of guinea-pig trachealis which was unaffected by atropine (1 mumoll-1), mepyramine (1 mumoll-1), tetrodotoxin (3 mumoll-1) or indomethacin (2.8 mumoll-1). Spasm evoked by KC1 was depressed in Ca2+-free Krebs solution or by exposure of tissues to LaCl3 (0.25-1 mmoll-1). Extracellular electrical recording showed that the spasm evoked by KCl 10 mmoll-1 was associated with promotion of electrical slow wave activity. Higher concentrations of KC1 abolished slow wave activity but caused further tension development. Intracellular recording confirmed the ability of KC1 10 mmoll-1 transiently to promote slow wave activity in individual trachealis cells. This action was associated with depolarization and tension development. Higher concentrations of KC1 evoked further tension development but slow waves were suppressed as the depolarization evoked by KC1 increased. KC1 (10-40 mmoll-1) increased the lanthanum-resistant calcium fraction of muscle-containing strips of trachea. It is concluded that KC1 acts directly on the smooth muscle of guinea-pig trachea. The spasmogenic action is associated with transient promotion of slow wave activity and a fall in resting membrane potential. The spasm involves the cellular influx of Ca2+ and is dependent on the presence of Ca2+ in the extracellular fluid.

Electron microscopy showed that the process of skinning guinea pig trachaelis muscle using Triton X-100 destroys the plasma membranes and causes some retraction of the myofibrils toward the center of the cells. Seven days represents an optimal period for the low temperature storage of the skinned fibers. Increasing the imposed tension from 0.25 to 1.0 g reduces the size of the maximal response to Ca2+. Some sensitization of the skinned fibers to Ca2+ follows the construction of the initial Ca2+ log concentration-response curve. Exogenous calmodulin is not essential for Ca2+-induced tension development and, at 20 degrees C, does not potentiate Ca2+. Useful economy can be achieved without compromising Ca2+ sensitivity or responsiveness by using ATP (a component of the relaxing solution) of purity 99% rather than of 99-100%.

The properties and physiological roles of K+-channels in the plasmalemma of airways smooth muscle are currently being clarified by the use of techniques such as patch-clamp recording. K+-channel activity plays an important role in determining both the resting membrane potential and the electrical excitability of the airways smooth muscle cell. Established bronchodilators such as agonists at beta-adrenoceptors and methylxanthines may provide K+-channel opening but this is not crucial for their relaxant action. The actions of other smooth muscle relaxants such as BRL 34915 and pinacidil (Fig.) may more closely depend on their ability to open K+-channels and such agents are currently being examined to determine whether they protect against bronchoconstrictor challenge in vivo or have beneficial effects in asthma.

A low concentration (0.2 nM) of oxytocin induced phasic tension development in the isolated uterus of the day-22 pregnant rat. Tonic spasm was also induced by higher concentrations of oxytocin (2 and 20 nM). Spasmogenic responses to bradykinin and potassium chloride (KCl) also contained phasic and tonic components while acetylcholine induced tonic spasm only. The phasic component of the responses to oxytocin and to bradykinin and both components of the response to KCl were inhibited by (+)-cis diltiazem (0.1 and 1 microM). The tonic component of the responses to oxytocin and to bradykinin and the responses to acetylcholine were only reduced by (+)-cis diltiazem at concentrations greater than 10 microM. (-)-cis Diltiazem was less potent than (+)-cis diltiazem as an inhibitor of calcium (Ca2+)-induced spasm in a depolarizing medium and of the phasic spasms induced by oxytocin. The two isomers were of similar potency as inhibitors of oxytocin-induced tonic spasm. Spasmogenic responses to oxytocin, bradykinin, acetylcholine and KCl were decreased when uteri were bathed in media which were Ca2+-free or of low Na+ content. However, there was no correlation between the rank order of sensitivity of the four spasmogens to the changed media and to their inhibition by (+)-cis diltiazem. Oxytocin (0.2 nM) increased the frequency, duration and amplitude of spike activity, measured by extracellular electrical recording, in parallel with enhancement of phasic tension development. With higher concentrations of oxytocin (2 and 20 nM) spike firing was initially continuous but often subsequently ceased despite the associated tonic contracture. After incubation in (+)-cis diltiazem (10 microM), oxytocin (0.2, 2 and 20 nM) produced graded tonic spasm without spike activity. Oxytocin (0.2 nM) produced a small increase in 45Ca2+ influx into myometrium as assessed by the 'lanthanum method'. Higher concentrations of oxytocin (2 and 20 nM) did not increase 45Ca2+ influx. It is concluded that the phasic component of the response of the uterus to oxytocin and bradykinin is associated with Ca2+ influx via voltage-dependent Ca2+ channels. The tonic component is due to another mechanism(s) which does not appear to involve Ca2+ influx. All of the spasmogenic response to KCl can be explained by Ca2+ influx through voltage-dependent Ca2+ channels. These channels do not appear to be involved in the spasmogenic response to acetylcholine.

1. Caffeine (10 mM)-induced relaxation of guinea-pig isolated trachealis was attenuated and converted to a small spasmogenic response on cooling to 22 degrees C. The relaxant response was restored on rewarming to 37 degrees C and was abolished by indomethacin (2.8 microM). Cooling to 22 degrees C in the presence of indomethacin revealed spasmogenic responses to caffeine which were abolished on rewarming to 37 degrees C. 2. Trachealis treated with indomethacin (2.8 microM) was repeatedly dosed with acetylcholine (ACh, 10 microM). Caffeine (1 or 10 mM), added as each ACh-induced spasm reached equilibrium, transiently augmented but then suppressed the spasm. On cooling from 37 degrees C to 12 degrees C, the increment in spasm evoked by caffeine increased relative to the spasm evoked by ACh. 3. Trachealis treated with indomethacin (2.8 microM) was repeatedly dosed with caffeine (10 mM). At 37 degrees C caffeine had little effect but it caused spasm when the tissue was cooled to 32 degrees C. Spasm amplitude increased as cooling progressed to 12 degrees C. Similar results were obtained with caffeine (1 mM). 4. At 37 degrees C, caffeine, enprofylline, 1,3,7,9-tetramethylxanthinium (TMX), theobromine, theophylline, xanthine and forskolin each caused concentration-dependent suppression of tracheal tone. Among the xanthine derivatives the rank order of potency was enprofylline greater than theophylline greater than caffeine greater than theobromine greater than xanthine greater than TMX. 5. In trachealis treated with indomethacin (2.8 microM) and maintained at 12 degrees C, the xanthines each caused concentration-dependent spasm. The rank order of potency was theobromine greater than or equal to theophylline greater than or equal to caffeine greater than or equal to enprofylline greater than xanthine greater than TMX. Forskolin was devoid of spasmogenic activity. 6. Trachealis treated with indomethacin (2.8 microM) and maintained at 12 degrees C, was repeatedly dosed with either caffeine (10 mM) or potassium chloride (KCl, 40 mM). Caffeine-induced spasm was attenuated in a Ca2+-free medium containing EGTA (2 mM), modestly at first but subsequently more profoundly. KCl did not evoke spasm at 12 degrees C but at 37 degrees C the KCl-induced spasm was virtually abolished at its first trail in the Ca2+-free, EGTA-containing medium. 7. It is concluded that caffeine, other alkylated xanthines and xanthine itself share a spasmogenic action in guinea-pig isolated trachealis which is best observed when the tissue is treated with indomethacin (2.8 microM) and maintained at 12 degrees C. The spasmogenic action represents the release of Ca2+ from intracellular sites of sequestration and may not depend on the intracellular accumulation of cyclic AMP. The rank order of spasmogenic potency of the xanthine derivatives differs markedly from their rank order of potency in suppressing the spontaneous tone of the trachealis observed at 370C. Since, at 12 degrees C, TMX is spasmogenic at concentrations identical to those causing relaxation at 37 degrees C, it is likely that TMX penetrates the cell. The relaxant effects of TMX do not, therefore, indicate that methylxanthine-induced relaxation is mediated by a receptor located on the external surface of the cell.

Radiation therapy to either mediastinum or pelvis causes a rapid decrease in circulating lymphocytes of both B and T types and in addition an impairment in the function of the remaining lyphocytes, as measured by their ability to proliferate in response to mitogens. The acute depression is short-lived. Substantial recovery is apparent within 3 wk after cessation of therapy; however, most patients show a modest, chronic depression in both numbers and functional capacities of circulating lymphocytes. T cells are somewhat more sensitive than B cells, but both are affected. Irradiation of the thymus per se seems to have little influence on the acute changes which occur, as patients receiving pelvic and mediastinal (including thymic) radiotherapy show a similiar degree of lymphopenia and depression of lymphocyte responsiveness.

1 Hyoscine- and guanethidine-treated preparations of longitudinal muscle of rabbit duodenum, guinea-pig taenia caeci and fundic strip relaxed when exposed to noradrenaline, adenosine, adenosine triphosphate (ATP) or to field stimulation of their intramural nerves. 2 In guinea-pig taenia caeci and fundus, theophylline 100 mumol/l had no effect on responses to noradrenaline, adenosine, ATP and intramural nerve stimulation. 3 In rabbit duodenum, theophylline 100 mumol/l antagonized some responses to adenosine but had no effect on responses to noradrenaline, ATP and intramural nerve stimulation. 4 Theophylline 1 mmol/l itself relaxed the intestinal tissues and in the fundic strip and taenia caeci, these relaxant effects were associated with abolition of spike activity and cellular hyperpolarization. In the taenia caeci, the amplitude of inhibitory post-junctional potentials was reduced. 5 Theophylline 1 mmol/l antagonized the twitch suppression produced by adenosine and ATP in the transmurally-stimulated guinea-pig ileum but not that evoked by noradrenaline. 6 It is concluded that theophylline can selectively antagonize some actions of purines in the intestine but that it does not specifically antagonize the effect of intramural inhibitory nerve stimulation.

In trachealis bathed by a K+-rich, Ca2+-free physiological salt solution, calcium chloride (CaCl2) at 0.01 to 10 mmol l-1 evoked concentration-dependent spasm. Verapamil (0.1 to 10 mumol l-1) was an effective antagonist of CaCl2. Spasm evoked by acetylcholine, histamine, potassium chloride (KCl) and tetraethylammonium (TEA) was studied in trachealis bathed by normal Krebs solution. Verapamil (0.1 to 10 mumol l-1) markedly suppressed spasm evoked by KCl and TEA. In contrast the actions of acetylcholine and histamine were much less affected by verapamil. Spasm evoked by prostaglandin E2 was studied in trachealis bathed by Krebs solution containing indomethacin (2.8 mumol l-1). Verapamil (0.1 to 10 mumol l-1) had little or no effect against prostaglandin E2-induced spasm. Verapamil (0.1 to 10 mumol l-1) had relatively little effect on the tone of trachealis bathed by normal Krebs solution. In contrast bathing in Krebs solution lacking CaCl2 caused almost complete tone loss. Extracellular electrophysiological recording showed that verapamil (10 mumol l-1) suppressed not only TEA-evoked spasm but also TEA-evoked slow waves and spike potentials. Verapamil also abolished the transient period of slow wave activity associated with the spasm evoked by KCl. Intracellular electrophysiological recording showed that TEA-induced spike activity was resistant to tetrodotoxin (3 mumol l-1). However, verapamil (10 mumol l-1) abolished the tetrodotoxin-resistant spikes without increasing the resting membrane potential. It is concluded that verapamil suppresses TEA- or KCl-induced spasm, slow waves or spikes by inhibition of Ca2+ influx. Spasm evoked by acetylcholine, histamine and prostaglandin E2 depends on mechanisms for increasing the cytoplasmic concentration of free Ca2+ which are resistant to verapamil. The failure of verapamil markedly to depress tissue tone is consistent with the proposal that tone results from the activity of endogenous prostaglandins.

Intracellular electrophysiological recording showed that acetylcholine (1 mumol l-1) and histamine (2 mumol l-1) depolarized trachealis cells and often increased the frequency of slow waves. Higher concentrations of these agents caused greater depolarization and abolition of slow waves. Marked depolarization was often associated with the appearance of electrical 'noise'. These electrical phenomena were accompanied by tonic tension development in a contiguous segment of trachea. Electrical 'noise' and tension evoked by high concentrations of acetylcholine or histamine could be dissipated by washing the agonist from the tissue. Acetylcholine-induced 'noise' was resistant to tetrodotoxin (3 mumol l-1) and to hexamethonium (1 mmol l-1). Neither acetylcholine (10-1,000 mumol l-1) nor histamine (2-200 mumol l-1) increased the lanthanum-resistant calcium fraction of muscle-containing strips of trachea. It is concluded that, while developing tension under the influence of acetylcholine or histamine, trachealis cells depolarize markedly but there is relatively little cellular influx of Ca2+.

1. Acetylcholine (ACh), histamine, prostaglandin E2 and potassium chloride (KCl) each evoked concentration-dependent spasm of guinea-pig isolated trachealis treated with indomethacin (2.8 microM). 2. Neither tetraethylammonium (TEA; 0.1-10 mM) nor procaine (0.1-10 mM) potentiated these spasmogens. Indeed, procaine (10 mM) depressed the log concentration-effect curves of all the spasmogens while TEA (1-10 mM) caused some depression of the log concentration-effect curve of prostaglandin E2. 3. Intracellular electrophysiological recording was performed in trachealis bathed by normal Krebs solution or by Krebs solution containing 2.8 microM indomethacin. In either medium the majority of trachealis cells exhibited spontaneous electrical slow waves while some cells were electrically quiescent. In either medium the spasmogenic effects of ACh (1 mM) and histamine (0.2 mM) were accompanied by depolarization and abolition of slow wave discharge. In many cases the record of membrane potential subsequently exhibited noise which incorporated fast, hyperpolarizing transients. 4. In the absence and presence of indomethacin, TEA (10 mM) and procaine (5 mM) markedly reduced the membrane noise and hyperpolarizing transients evoked by ACh or histamine without augmenting the evoked tension. 5. It is concluded that slow wave discharge does not depend on prostaglandin synthesis. The membrane noise and hyperpolarizing transients evoked by ACh and histamine represent the opening of membrane K+-channels. While such K+-channel opening may offset spasmogen-induced depolarization it does not moderate the evoked tension.

Nicorandil (1-1000 mumol l-1) caused concentration-dependent relaxation of guinea-pig isolated trachealis. Propranolol (1 mumol l-1) did not modify the relaxant action of nicorandil but antagonized isoprenaline. Among K+-channel inhibitors tested, apamin (0.1 mumol l-1) and procaine (5 mmol l-1) did not modify the relaxant action of nicorandil. In contrast, tetraethylammonium (TEA, 8 mmol l-1) caused five fold antagonism. Trachealis exposed to K+-rich (120 mmol l-1) Krebs solution developed near-maximal tension. Nicorandil relaxed the K+-depolarized tissue though its concentration-effect curve was shifted markedly to the right. In tissues in which tone was induced by histamine, methylene blue (100 mumol l-1) antagonized nicorandil and sodium nitroprusside but did not modify the relaxant action of aminophylline. Intracellular electrophysiological recording showed that nicorandil (1 mumol l-1) could evoke some relaxation in the absence of electrical changes. Higher concentrations (10-1000 mumol l-1) reduced the amplitude and frequency of spontaneous electrical slow waves. Nicorandil also caused concentration-dependent hyperpolarization and relaxation. When the hyperpolarization was sufficiently pronounced slow wave activity was abolished. TEA (8 mmol l-1) induced slow waves which were surmounted by a spike potential. TEA slightly reduced the maximal hyperpolarization induced by nicorandil and increased the time required for nicorandil to abolish slow wave discharge. Procaine (5 mmol l-1) induced slow waves of relatively low frequency. Sometimes these were surmounted by a spike potential Procaine markedly reduced the hyperpolarization induced by nicorandil and increased the time required for abolition of slow waves. In studies of the efflux of 86Rb+ from muscle-rich strips of trachea, nicorandil (1000 mumol l-1) increased the efflux rate constant, whereas isoprenaline (1 mumol l-1) was without effect. It is concluded that nicorandil-induced relaxation does not involve the activation of beta-adrenoceptors but is partly attributable to the formation of nitric oxide from the nitrate moiety in its molecular structure. Nicorandil can evoke relaxation in the absence of membrane potential change but towards the upper end of its effective concentration range, nicorandil increases membrane K+ conductance and thereby evokes hyperpolarization of trachealis cells. The K+ channels opened by nicorandil are permeable to 86Rb, insensitive to apamin and TEA but may be inhibited by procaine.

1. Experiments have been performed with the dual intent of analysing the mechanism by which AH 21-132 relaxes airways smooth muscle and determining whether the effects of this compound can be distinguished from those of theophylline. 2. AH 21-132 (0.25-8 microM) and theophylline (1-1000 microM) each caused concentration-dependent suppression of the spontaneous tone of guinea-pig isolated trachealis. The maximal effect of AH 21-132 was equivalent to that of theophylline. No evidence was obtained that the tissue became sensitized or desensitized to the action of AH 21-132. 3. Propranolol (1 microM) profoundly antagonized the tracheal relaxant action of isoprenaline but not that of AH 21-132. 4. In indomethacin (2.8 microM)-treated tissues, tone was induced by K+-rich (120 mM) Krebs solution, acetylcholine (ACh, 1 mM) or histamine (200 microM). Log concentration-relaxation curves for AH 21-132, isoprenaline and theophylline were all moved to the right in the presence of the spasmogens, the smallest rightward shift being induced by histamine and the greatest by ACh. While maximal effects of AH 21-132 and theophylline were unaffected by the spasmogens, that of isoprenaline was reduced by KCl and ACh. 5. In tissues treated with indomethacin (2.8 microM), AH 21-132 (0.1-100 microM) inhibited the spasmogenic effects of potassium chloride (KCl), ACh and histamine in a concentration-dependent manner. The inhibition was characterized by rightward shifts in the spasmogen concentration-effect curves with depression of their maxima. 6. In tissues treated with both indomethacin (2.8 microM) and ACh (1 mM), the removal of tracheal epithelium caused a small but significant leftward shift in the log concentration-relaxation curve for AH 21-132 but did not alter that for theophylline. 7. In tissues treated with indomethacin (2.8 microM) and maintained at 12 degrees C, theophylline (0.1-3.2 mM) caused concentration-dependent spasm. This effect was not shared by AH 21-132. 8. AH 21-132 (0.1-1000 microM) more potently inhibited the activity of cyclic AMP-dependent than of cyclic GMP-dependent phosphodiesterase derived from homogenates of guinea-pig trachealis. Theophylline, too, inhibited these enzymes but was less potent in each case than AH 21-132 and did not exhibit selectivity for the cyclic AMP-dependent enzyme. 9. It is concluded that AH 21-132 exerts a non-specific (i.e. effective no matter what agent is used to support tone) relaxant effect on the trachealis muscle which does not involve the activation of beta l-adrenoceptors. The profile of the relaxant action of AH 21-132 more closely resembles that of theophylline than that of isoprenaline. However, AH 21-132 can be differentiated from theophylline in that: (a) its relaxant potency is increased by epithelial removal; (b) it does not cause tracheal spasm; (c) it exhibits selectivity as an inhibitor of cyclic AMP-dependent as opposed to cyclic GMP-dependent phosphodiesterase. It is possible that the relaxant effects of AH 21-132 are related to its ability to inhibit cyclic nucleotide phosphodiesterases.

1 BRL 34915 (0.04-1.3 microM) caused concentration-dependent inhibition of spontaneous phasic spasms of the isolated uterus of the term pregnant rat and this effect was not antagonized by propranolol. Spasms evoked by low concentrations of KCl (less than 20 mM) were inhibited by BRL 34915 but those evoked by higher concentrations (greater than 40 mM) were unaffected. 2 In experiments using extracellular electrical recording, BRL 34915 (10 microM) selectively inhibited oxytocin-induced phasic spasms and the associated spike activity but had little effect on the tonic component of the spasms. BRL 34915, as an inhibitor of phasic spasms to oxytocin (0.2 nM), was antagonized by procaine (0.3 and 1 mM). 3 BRL 34915 (10 microM) did not inhibit Ca2+-induced spasm of saponin-skinned thin myometrial strips. 4 Intracellular microelectrode recording from myometrial strips showed that BRL 34915 (10 microM) inhibited action potentials and phasic spasms in the presence of oxytocin (0.2 nM) and produced a hyperpolarization of 5 mV. 5 In single myometrial cells under current or voltage clamp, BRL 34915 (10 microM) had no effect on action potentials and inward current in Ca2+- or Ba2+-containing media in the presence of tetraethylammonium, 4-aminopyridine and caesium chloride. In the absence of these K+-channel inhibitors, BRL 34915 had no effect on resting membrane potential, membrane resistance, action potential, inward current or outward current. 6 BRL 34915 (1 or 10 microM) had no effect on 86Rb efflux from myometrial strips. 86Rb efflux was increased by oxytocin (0.2 and 20 nM). 7 The relaxant profile of BRL 34915 in the rat uterus is similar to that described for other smooth muscles where an action to open membrane K+-channels has been proposed. BRL 34915 inhibited spike production but produced only a small hyperpolarization without a detectable increase in 86Rb efflux. Membrane resistance and transmembrane currents were unaffected. These results suggest that in the uterus the effects of BRL 34915 may be restricted to K+-channels involved in the production of pacemaker activity.